Current methods of purifying a naturally-occurring or recombinant therapeutic protein from a solution comprising said protein usually carry at least some impurities into the final preparation. In some instances, the presence of impurities, such as proteases, will destabilise the therapeutic protein in solution, particularly during storage. For this reason, many therapeutic proteins are stored as lyophilized or frozen preparations.
Whilst destabilising levels of impurities can affect many different types of therapeutic proteins, they are particularly relevant to those used for maintaining haemostasis. Haemostasis is an important physiological process that prevents bleeding following damage (e.g. a rupture) to blood vessels. There are three basic mechanisms that promote haemostasis: (i) vasoconstriction, (ii) platelet aggregation at the rupture site; and (iii) coagulation. During coagulation, damaged endothelial cells release tissue factor (Factor III), which in turn activates Factor VII with the aid of Ca2+. Factor XII, which is released by activated platelets, activates Factor XI. Activated Factor VII and Factor XI promote a cascade of enzymatic reactions that lead to the activation of Factor X. Active Factor X (Factor Xa), along with Factor III, Factor V, Ca2+, and platelet thromboplastic factor (PF3), activate prothrombin activator. Prothrombin activator converts prothrombin to thrombin, which converts fibrinogen (Factor I) to fibrin, which forms an initial mesh over the site of damage. The initial mesh is then converted to a dense fibrin clot by Factor XIII, sealing the rupture until the site is repaired. During the coagulation cascade, thrombin will also activate Factor VIII, a glycoprotein pro-cofactor that in the circulation is mainly complexed to von Willebrand factor (VWF). Factor VIII interacts with Factor IXa to activate Factor X in the presence of Ca+2 and phospholipids.
A deficiency in the level of any one or more of the proteins involved in coagulation, including fibrinogen, Factor VIII, and/or von Willebrand factor (VWF) whether congenital or acquired, can lead to insufficient clotting of blood and the risk of haemorrhage. Current treatment options are limited to the administration of a pharmaceutical preparation of one or more therapeutic proteins, with a view to restoring endogenous levels of said proteins and maintaining haemostasis. However, existing pharmaceutical preparations, which are typically derived from donated blood plasma or a recombinant source, comprise zymogens and proteases (e.g., prothrombin, plasminogen, tissue plasminogen activator (tPA) and/or other proteases), which can destabilise the therapeutic proteins, such as fibrinogen, Factor VIII, or VWF during storage. As a consequence, such preparations are relatively unstable in aqueous solution, with long-term storage limited to lyophilized or frozen preparations.
For clinical applications, fibrinogen is typically purified from human plasma, where it accounts for only about 2-5% (1.5-4.0 g/L) of total plasma proteins. Traditionally, the purification of fibrinogen from plasma is carried out by classical plasma fractionation, where fibrinogen is cryo-precipitated from plasma followed by precipitation with either ethanol, ammonium sulphate, β alanine/glycine, polymers (e.g., polyethelene glycol) or low ionic strength solutions. Such methods can achieve relative high yield and homogeneity. Where a greater level of purity is required, chromatographic techniques are often employed. However, existing precipitation and chromatographic techniques amenable to commercial scale manufacturing processes typically produce fibrinogen preparations that comprise contaminating proteins such as zymogens or proteases (e.g., prothrombin, tissue plasminogen activator (tPA) and plasminogen), which can destabilize fibrinogen in solution. For example, when prothrombin is present, it can be activated to the serine protease thrombin which will in turn convert fibrinogen into fibrin. Similarly, when both tPA and plasminogen are present, tPA can activate plasminogen to its active form plasmin, which will in turn hydrolyse fibrinogen into fibrin. As a consequence, fibrinogen preparations are relatively unstable in aqueous solution, with long-term storage limited to lyophilized or frozen preparations.
Specific contaminants can be absorbed out; for example, fibronectin on immobilised gelatine and plasminogen on immobilised lysine (Vuento et al. 1979, Biochem. J., 183(2):331-337). However, the use of specific affinity resins is not amenable to large scale commercial processes. Reasons for this include the affinity resins themselves not being sufficiently robust to be used repeatedly and generally add significantly to the both processing time and costs.
EP1240200 (U.S. Pat. No. 6,960,463) is directed to methods of purifying fibrinogen from a fibrinogen-containing solution using ion exchange (IEX) chromatography. In particular the method involves applying a fibrinogen-containing solution to an ion exchange matrix under conditions that allow the fibrinogen to bind to the matrix and then washing the ion exchange matrix with a solution comprising at least one omega amino acid. This is done to promote the differential removal of plasminogen from the resin. Fibrinogen that is bound to the matrix is then eluted from the matrix.
WO2012038410 provides a method of purifying fibrinogen using anion exchange resins which contain a hydroxylated polymer support grafted with tertiary or quaternary amines that bind fibrinogen.
EP519944 teaches the use of an immobilized metal ion affinity chromatography matrix under conditions that fibrinogen and plasminogen bind to the matrix, and selectively eluting the fibrinogen and plasminogen separately, such that the main fibrinogen fraction contains about 600 ng of plasminogen per mg of protein.
The present invention provides a method of reducing the level of plasminogen and/or tissue plasminogen activator and/or other protease(s) in a solution comprising fibrinogen and/or Factor VIII and/or VWF. The purified protein(s) are stable during storage as liquid preparations and can be used for clinical or veterinary applications, including treating or preventing conditions associated with a deficiency in the level of said protein(s).